Antibody antagonists of ve-cadherin without adverse effects on vascular permeability

ABSTRACT

The murine epitope sequence recognized by antibody E4B9 shares 100% homology with human VE-cadherin, so this antibody was examined to determine if it cross-reacts with human VE-cadherin. Western-blot analysis of several VE-cadherin expressing human and murine cells indicated that E4B9 indeed cross-reacts with human VE-cadherin (FIG.  6 ). This finding facilitates development of a “humanized” E4B9 antibody and its success in the preclinical development since its anti-tumor activity can be tested extensively in several mouse models.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 10/040,128 filed Jan. 2, 2002, which is acontinuation of U.S. application Ser. No. 09/540,967 filed Mar. 31,2000, now abandoned, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to antibody antagonists of VE-cadherinthat inhibit formation new of adherens junctions without disrupting theintegrity of existing junctions. Such antibodies are useful to preventangiogenesis in a variety of disease conditions, including, for example,to prevent neovascularization of tumors. These antibodies are alsouseful for treating endothelial cell proliferative disorders.

BACKGROUND OF THE INVENTION

Many diseases are associated with an abnormal proliferation of bloodvessels. The process of forming new blood vessels is termedangiogenesis. Under normal or non-pathologic conditions angiogenesisoccurs under well-defined conditions such as in wound healing, inresponse to ischemia and during embryonal and fetal development.However, persistent or uncontrolled angiogenesis can lead to a varietyof disease states or conditions and, in the case of solid tumors, may bea necessary condition to maintain the disease state. For example,angiogenesis occurs with neoplastic diseases, particularly with solidtumors, in autoimmune diseases, in collagenous vascular diseases such asrheumatoid arthritis, and in certain ophthalmalogical conditions such asdiabetic retinopathy, retrolental fibroplasia and neovascular glaucoma.One therapeutic approach for the treatment of such diseases would be torestrict, reduce or eliminate the blood supply to the diseased cells ortissues. For example, solid tumors greater than a few millimetersundergo neovascularization without which further tumor growth would beimpossible, so that inhibiting blood vessel formation will limit tumorsize.

Some treatment strategies have attempted to limit the tumor's bloodsupply by occluding blood vessels supplying the tumor. For suchtreatment, the site of the tumor must be known and the tumor must beaccessible. Thus a method of treatment that did not rely on knowing thelocation of or the accessibility to the site of interest would bevaluable and could permit systemic delivery of a therapeuticanti-angiogenesis agent capable of specifically targeting a diseasesite.

Because of the role that angiogenesis plays in the development ofdisease, there is substantial interest in the development ofangiogenesis inhibitors, especially where current therapies are lessthan optimal. Since endothelial cells are an integral part of bloodvessel formation, a specific inhibitor of such cells would beadvantageous in inhibiting angiogenesis, provided, of course, there is aminimum toxicity associated with that inhibitor. One particular targetof interest is the endothelial cell-specific cadherin, VE-cadherin, thatforms intercellular adherens junctions.

Cadherins are a family of cell adhesion molecules that are involved inthe formation of specific cell-cell contacts (Takeichi, Ann. Rev.Biochem. 59: 237-252 (1990); Geiger & Ayalon, Ann. Rev. Cell Biol. 8:302-332 (1992); Uemura, Cell 93: 1095-1098 (1998)). A number of membershave been identified or characterized. Cadherins are single chaintransmembrane glycoproteins with molecular weights of 120-140 kD.Members of this family exhibit calcium-dependent homophilic interactionsand are responsible for the selective cell-cell recognition andadhesion, which is necessary for allocating different cell types totheir proper places during organ development. Cadherins also play animportant role in maintaining the integrity of multicellular structures.During embryonic morphogenesis the expression of diverse members of thecadherin family is spatially and temporally regulated facilitating theorderly assembly of various cell types into functional structures(Takeichi, Ann. Rev. Biochem. 59: 237-252 (1990)).

Members of the cadherins family have typical structural features andshare considerable sequence homology (43-58%). Their extracellularregion typically contains 5 repeating domains of approximately 110 aminoacids. The N-terminal domain has been shown to be important in homotypiccell-cell interaction as evidenced by experiments with molecularchimeras, monoclonal antibodies and peptide inhibitors (Nose et al.,Cell 54: 993-1001 (1988)). The 3-dimensional structures of theN-terminal domains of N-cadherin and E-cadherin have been elucidated(Shapiro et al., Nature 374: 327-337 (1995); Overduin et al., Science267: 386-389 (1995); Nagar et al., Nature 380: 360-364 (1996)).Accordingly, it is believed that cadherins form dimers supported byzipper-like elements and possibly by disulfide linkage. The shortintracellular portion of cadherins is their most highly conserved regionand plays an essential role in classic cadherin function by anchoringcadherins to the cytoskeleton and providing signaling functions throughcadherin phosphorylation (See, FIG. 1).

VE-cadherin (or cadherin-5) has been shown to be localized atintercellular junctions (adherens junctions) in cell-to-cell contacts(Lampugnani et al., J. Cell. Biol. 118: 1511-1522 (1992); Breviario etal., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239 (1995); Breier etal., Blood 87: 630-641 (1996); Lampugnani et al., J. Cell Biol. 129:203-217 (1995)). A number of experimental observations suggest that thiscadherin is involved in various aspects of vascular biology related toangiogenesis, including the assembly of endothelial cells into tubularstructures (Bach et al., Experimental Cell Research 238: 324-334(1998)). For example, thrombin-induced vascular permeability is shown tobe associated with disassembly of endothelial adherens junctions (Rabietet al., Arterioscler. Thromb. Vasc. Biol. 16: 488-496 (1996); Dejana, J.Clin Invest. 100: S7-10. (1997); Dejana et al., FASEB J., 9: 910-918(1995); Dejana et al., Ann N Y Acad Sci. 811: 36-43 (1997); Gotsch etal., J. Cell. Sci. 110: 583-588 (1997); Kevil et al., J. Biol. Chem.273: 15099-15103 (1998); Corada et al., Proc. Natl. Acad. Sci. 96:9815-9820 (1999)). VE-cadherin and its N-terminal fragment inhibit thedensity-dependent growth (Yap et al., J. Cell Biol. 141: 779-789 (1998);Caveda et al., J. Clin. Invest. 98: 886-893 (1996)) and migration(Breviario et al., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239(1995)) of endothelial cells. In other experiments, VE-cadherin wasshown to confer adhesive properties to transfected cells (Breviario etal., Arterioscler. Thromb. Vasc. Biol. 15: 1229-1239 (1995); Breier etal., Blood 87: 630-641 (1996); Ali et al., Microcirculation 4: 267-277(1997)), and an essential role for VE-cadherin in blood vessel formationhas been demonstrated in VE-cadherin null mice. In these mice, severelyimpaired assembly of vascular structures leads to an embryonic lethalphenotype (Vittet et al., Proc. Natl. Acad. Sci. 94: 6273-6278 (1997);Faure et al., Development 128: 2093-2102 (1999); Carmeliet et al., Cell98: 147-157 (1999)). These findings strongly validate VE-cadherin as anattractive pharmacological target for inhibiting neovascularization.

Prior to the present invention, attempts to use VE-cadherin antibodyantagonists to prevent angiogenesis have been limited by the toxicity ofthe antibody to normal vasculature. For example, administering certainanti-cadherin antibodies in amounts sufficient to prevent or inhibitangiogenesis have resulted in disturbances in the integrity of normalvasculature with resultant vascular leak syndromes, hemorrhage anddeath. For example, the anti-VE-cadherin antibody 19E6 results inincreased pulmonary vascular permeability because that antibody disruptsexisting VE-cadherin-mediated cellular junctions as well as preventingformation of new VE-cadherin-mediated cellular adherens junctions. Thepresent invention addresses now provides improved VE-cadherin antibodyantagonists directed to particular sites on VE-cadherin and whichovercome such problems.

SUMMARY OF THE INVENTION

The present invention is directed to an antibody or an antibody fragmentthat is an antagonist of VE-cadherin. The antibody and antibodyfragments of the invention are capable of specifically binding to amolecule selected from the group consisting of

a site on a VE-cadherin, said site being within the about 15 to about 20N-terminal amino acids of domain 1 of a VE-cadherin,

a site on a VE-cadherin, said site being within the about 15 to about 20N-terminal amino acids of domain 1 of a VE-cadherin and said N-terminalamino acids having an insertion, deletion or substitution of from 1 toabout 5 amino acids relative to a native VE-cadherin amino acidsequence,

a peptide having an amino acid sequence of SEQ ID NO: 1(DEIWNQMHIDEEKNE),

a peptide having an amino acid sequence of SEQ ID NO: 2(DWIWNQMHIDEEKNE), and

a peptide having an amino acid sequence of SEQ ID NO: 3(DWIWNQMHIDEEKNT). Furthermore, the antibody or antibody fragment of theinvention is capable of inhibiting VE-cadherin mediated adherensjunction formation in vitro but does not exert any significant orsubstantial effect on paracellular permeability in vitro. Suchantibodies and antibody fragments do not exert any significant orsubstantial effect on vascular permeability in vivo and aresubstantially non-toxic when administered to an animal or mammal. Inaddition, the antibodies or antibody fragments are capable of inhibitingangiogenesis in vivo or in vitro as well as tumor metastasis. Theantibodies and antibody fragments of the invention act by inhibitingformation of new adherens junctions without disturbing existing adherensjunctions. Preferred antibodies of the invention are monoclonalantibodies. Likewise, preferred antibody fragments are from monoclonalantibodies. In a more preferred embodiment, the monoclonal antibody ismonoclonal antibody E4B9. The preferred mammal of the invention is ahuman.

The antibodies and or antibody fragment of the instant invention can bea single chain antibody, humanized, chimerized, bispecific, or fused toa heterologous polypeptide.

Another aspect of the invention is directed to a hybridoma whichproduces the monoclonal antibodies of the invention.

A further aspect of the invention provides pharmaceutical compositionscomprising the antibody or antibody fragment of the invention inadmixture with a pharmaceutically acceptable carrier or diluent.

Yet another aspect of the invention relates to a method of inhibitingangiogenesis in a mammal by administering the pharmaceutical compositionof the invention to a mammal for a time and in an amount effective toinhibit angiogenesis.

Still another aspect of the invention is directed to a method ofinhibiting tumor metastasis in a mammal by administering thepharmaceutical composition of the invention to a mammal for a time andin an amount effective to inhibit metastasis of a tumor.

Further still, the invention includes a method of treating a cellproliferative disorder associated with vascularization in a mammal byadministering a pharmaceutical composition of the invention to a mammalin an amount effective to inhibit proliferation of endothelial cellswithout disturbing the normal vasculature. Cell proliferative disorder,include but are not limited to, blood vessel proliferative disorders,fibrotic disorders, angiogenesis, tumor growth, tumor metastasis,rheumatoid arthritis, and age-related muscular degeneration.

Yet another embodiment of the invention provides a method for reducingor inhibiting tumor vasculature in a mammal by administering apharmaceutical composition of the invention to a mammal in an amounteffective to inhibit blood vessel formation without adversely affectingexisting vasculature, i.e., so to eliminate or substantially reduce orrestrict blood flow to a tumor without adversely affecting existingvasculature.

The invention also provides an isolated nucleic acid comprising anucleotide sequence which encodes a coding sequence for the antibody orantibody fragment, for a variable region of said antibody or for ahypervariable region of said antibody in accordance with the invention.

In yet a still further embodiment the present invention is directed to amethod of gene therapy to deliver the antibody or antibody fragment ofthe invention to a mammalian host. This method is comprisesadministering a nucleic acid encoding the desired antibody or antibodyfragment to a mammal in an amount and for a time effective to inhibitangiogenesis at a predetermined site or to inhibit tumorneovascularization.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: VE-cadherin Dimerization. Two forms of VE-cadherin dimers areproposed based on the crystal structures resolved for N- andE-cadherins. The “strand dimer” (left panels) refers to homophilicinteractions between two VE-cadherin molecules on the surface of thesame cell. The “adhesion dimer” (right panels) refers to homophilicinteractions between VE-cadherin molecules located on opposing cells.

FIG. 2: Sequence Alignment of ECD1 of Four Classic Cadherins. Fourregions of domain 1 for VE-cadherin are predicted to encompass thebinding surface of either the strand dimer or the adhesion dimer. Fourpeptides (lower panels) are synthesized that encompass these regions togenerate specific antibody inhibitors. Peptides 1: DEIWNQMHIDEEKNE-Cys;2: YVKDQSNYNRQNAKY-Cys; 3: KYVLQGEFAGKIFGVDA-Cys and 4:LIVDKNTNKNLEQP-Cys. These peptides are represented by SEQ ID NOS. 1 and4-6, respectively. The cysteine residue was added at the carboxyl end ofeach peptide for KLH-coupling.

FIG. 3: Effects of the anti-ECD1 (extracellular domain) peptidesantibodies on paracellular permeability of H5V cells.

FIG. 4: Antibody E4B9 does not exhibit significant effect onparacellular permeability. Antibodies E4B9 and 6D10 do not exertdramatic effect on vascular permeability.

FIGS. 5A & 5B: Antibody E4B9 exhibits potent anti-angiogenesis activityin mouse corneal micropocket assay. Three representative eyes from eachexperimental group (6 mice/group) are tested. Antibody E4B9possesses >80% inhibitory activity on corneal neovascularization.

FIG. 6: Antibody E4B9 cross-reacts with human VE-cadherin.

FIG. 7: Epitope mapping of new monoclonal antibodies. Strategy formapping the epitope of mAb 19E6 and 6D10.

FIG. 8: Summary of the epitope information for anti-ECD1 peptideantibodies. Antibody 10G4 epitope was mapped to the domain 1 of mouseVE-cadherin using the same strategy as previously described in FIG. 7.

FIG. 9: Predicted epitope region for antibody 19E6 and 10G4. Theunderlined regions are the epitopes for antibodies E4B9 and Cad-5,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provide antibody antagonists for VE-cadherin thatinhibit VE-cadherin to VE-cadherin interactions without substantiallydisrupting already formed adherens junctions. In so doing, theantagonist substantially inhibits or prevents intercellular formation ofnew adherens junctions without substantially disrupting existingadherens junctions. Thus, these antibodies, and fragments thereof thatretain the antigenic specificity of the intact antibody, are capable ofspecifically binding to a site on a mammalian VE-cadherin at the 15-20N-terminal amino acids of domain 1 of the mammalian VE-cadherin, arecapable of inhibiting VE-cadherin-mediated adherens junction formationin vitro but are not capable of exerting any significant or substantialeffect on paracellular permeability in vitro. The binding site ispreferably within the first 15 amino acids of the N-terminus of theVE-cadherin.

Alternatively, specific binding can be to a site on a mammalianVE-cadherin that is within the about 15 to about 20 N-terminal aminoacids of domain 1 of a VE-cadherin wherein the N-terminal amino acidshave an insertion, deletion or substitution of from 1 to about 5 aminoacids relative to a VE-cadherin amino acid sequence. Likewise, specificbinding can be to (1) a site with in the 15 N-terminal amino acids ofany allelic variation of a VE-cadherin; (2) a peptide having an aminoacid sequence of SEQ ID NO: 1 (DEIWNQMHIDEEKNE); a peptide having anamino acid sequence of SEQ ID NO: 2 (DWIWNQMHIDEEKNE); or a peptidehaving an amino acid sequence of SEQ ID NO: 3 (DWIWNQMHIDEEKNT). In allcases, the antibody antagonist retain the ability to inhibit formationof new junctions without disrupting existing junctions.

Hence the antibodies and antibody fragments of this invention do notexert any significant or substantial effect on vascular permeability invivo. Similarly, the antibodies and antibody fragments of the inventionare substantially non-toxic when administered to an animal or mammal.Likewise the antibodies and antibody fragments of the invention caninhibit angiogenesis in vivo or in vitro or inhibit tumor metastasis.The preferred antibody of the invention is murine monoclonal antibodyE4B9.

Mammals of the invention include, but are not limited to, domesticatedanimals (such as cattle, pigs, dogs and cats), mice, primates andhumans. Humans are the preferred mammal.

The antibodies and antibody fragments of the invention can be used to inmethods of inhibiting angiogenesis; in methods of inhibiting tumormetastasis; in methods of treating a cell proliferative disorderassociated with vascularization; and in methods for reducing orinhibiting tumor vasculature.

The present invention also includes chimeric, single chain, andhumanized antibodies, as well as diabodies, triabodies, Fab fragments,or the product of an Fab expression library.

The antibodies of the invention can be prepared by conventional methodswhich are well know in the art. Preferably the antibodies are monoclonalantibodies but the invention also contemplates use of monospecificpolyclonal antibodies. Monospecific polyclonal antibodies can beprepared by adsorbing out unwanted specificities from a preparation ofpolyclonal antibodies prepared with a suitable VE-cadherin immunogen.Immunogens suitable for preparation of the antibodies include but arenot limited to a mammalian VE-cadherin, fragments of a mammalianVE-cadherin, preferably extracellular domains from VE-cadherin, peptidesfrom the N-terminal domain 1 of a mammalian VE-cadherin, and fusionproteins with any of these molecules. Where appropriate the molecules(e.g., peptides) can be attached to carrier molecules such as BSA, KLHor any other carrier know in the art. The preferred immunogen is peptideconsisting essentially of the 15 N-terminal amino acid residues of amammalian VE-cadherin.

Techniques used for preparation of monoclonal antibodies, include butare not limited to, the hybridoma technique (Kohler & Milstein, Nature,256:495-497 (1975)), phage display techniques, the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72,(1983)), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole, et al., 1985, In Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96).

Another aspect of the invention includes the hybridomas which producemonoclonal antibodies of the invention. One such hybridoma producing ratanti-murine VE-cadherin E4B9 has been deposited with the American TypeCulture Collection (10801 University Blvd., Manassas, Va., 20110-2209USA (ATCC)) on Mar. 31, 2000, and assigned accession number PTA-1618.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778, incorporated herein by reference) are adapted toproduce single chain antibodies to immunogenic polypeptide products ofthis invention.

For example the antibodies of the invention can be raised againstVE-cadherin peptides, as well as fragments, analogs and derivatives of aVE-cadherin peptide. The terms protein, peptide, and polypeptide, areused interchangeably herein. The terms “fragment,” “derivative” and“analog” refer to a polypeptide which either retains substantially thesame biological function or activity as a VE-cadherin polypeptide, orretains the ability to bind the ligand even though the polypeptide doesnot function as a chemokine receptor, for example, a soluble form of themembrane polypeptide. The polypeptide of the present inventioncomprises, for example, a recombinant polypeptide, a natural polypeptideor a synthetic polypeptide.

An analog includes, for example, a proprotein which is activated bycleavage of the proprotein portion to produce an active maturepolypeptide. Fragments of VE-cadherin polypeptide include VE-cadherinpeptides having an N-terminal fragment comprising amino acid sequence ofFIG. 2, or a fragment thereof. Derivatives or analogs of the polypeptideof FIG. 2, include one or more sequences of SEQ ID NOS 1-3, andcomprise, for example, (i) peptides in which one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue, (ii) peptides in which one or more of the amino acidresidues include a substituent group, (iii) peptides in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), (iv) peptides in which the additional amino acids are fused tothe mature polypeptide for purification of the polypeptide(v) peptidesin which a fragment of the polypeptide is soluble, i.e. not membranebound, yet still binds ligands to the membrane bound peptide orreceptor, or (vi) a combination of (i) to (v). Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein. The polypeptides andpolynucleotides of the present invention are preferably provided in anisolated form, and preferably are purified to homogeneity. However, thisis not always necessary. Additionally, the polypeptides of the inventionhave at least 70% similarity (preferably a 70% identity) to one or morepeptides of SEQ ID NOS. 1-3 and more preferably a 90% similarity (morepreferably a 90% identity) to one or more peptides of SEQ ID NOs. 1-3and still more preferably a 95% similarity to the peptides of SEQ ID NOS1-3 and to portions of such peptide.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

According to another embodiment of the invention, the antibodies of theinvention can be prepared by recombinant DNA techniques by cloning andexpressing all or part of a known antibody. Using such techniques, whichare known in the art, a humanized version of non-human antibodies can beprepared for example a humanized version of monoclonal E4B9 can bereadily prepared by cloning the gene encoding this antibody in to anappropriate expression vector. Useful the nucleic acids in this regardare those which encodes an amino acid sequence wherein the amino acidsequence comprises the variable region, hypervariable region, or both ofa monoclonal antibody that specifically binds to domain 1 of anextracellular domain of a VE-cadherin peptide to inhibit new junctionformation without disturbing normal vasculature. More particularly, thepresent invention also includes recombinant constructs comprising one ormore of the sequences as broadly described above. The constructscomprise a vector, such as a plasmid or viral vector, into which asequence of the invention has been inserted, in a forward or reverseorientation. In a preferred embodiment of this embodiment, the constructfurther comprises regulatory sequences, including, for example, apromoter, operably linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

The constructs in host cells are used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).

According to another aspect of the invention, transgenic mammals areprovided that express humanized antibodies to immunogenic polypeptideproducts of this invention. Novel transgenic mammalian hosts, other thanprimates, particularly other than human, are provided, where the host iscapable of mounting an immune response to an immunogen, where theresponse produces antibodies having primate, particularly human,constant and/or variable regions or such other effector peptidesequences of interest.

The hosts are characterized by being capable of producing xenogeneic ormodified antibodies as a result of substitution and/or inactivation ofthe endogenous immunoglobulin subunit encoding loci. The modificationsretain at least a portion of the constant region which provides forassembly of the variable region binding site bonded at the C-terminus toa functional peptide. The functional peptide takes many forms orconformations and serves, for example, as an enzyme, growth factor,binding protein, ligand, cytokine, effector protein, chelating proteins,etc. The antibodies are any isotype, i.e., IgA, IgD, IgE, IgG, IgM orsubtypes within the isotype.

Transgenic hosts include murine, lagomorpha, ovine, porcine, equine,canine, feline, and the like. For the most part, mice have been used forthe production of B-lymphocytes. It should be understood that otheranimals may be readily substituted for the mice, following the sameprocedures.

Humanized and chimeric antibodies are prepared according to thefollowing strategies. In one strategy, the human heavy and light chainimmunoglobulin gene complexes are introduced into the mouse germ lineand in a separate step the corresponding mouse genes are renderednon-functional. Polynucleotides encoding human heavy and light chain arereconstructed in an appropriate eukaryotic or prokaryotic microorganismand the resulting polynucleotide fragments are then introduced intopronuclei of fertilized mouse oocytes or embryonic stem cells.Inactivation of the endogenous mouse immunoglobulin loci is achieved bytargeted disruption of the appropriate loci by homologous recombinationin mouse embryonic stem cells. In each case chimeric animals aregenerated which are derived in part from the modified embryonic stemcells and are capable of transmitting the genetic modifications throughthe germ line. The mating of mouse having a human immunoglobulin loci tomouse having an inactivated immunoglobulin loci yields animals thatproduce purely human antibody.

In another strategy, fragments of the human heavy and light chainimmunoglobulin loci are used to directly replace the corresponding mouseloci by homologous recombination in mouse embryonic stem cells. This isfollowed by the generation of chimeric transgenic animals. The resultinghuman antibodies are isolated, for example, from other proteins by usingan affinity column, having an Fc binding moiety, such as protein A, orthe like.

The organization, relative location of exons encoding individualdomains, and location of splice sites and transcriptional elements in anumber of animals are known by those of ordinary skill in the art. Inhuman, for example, the immunoglobulin heavy chain locus is located onchromosome 14. In the 5′-3′ direction of transcription, the locuscomprises a large cluster of variable region genes (V_(H)), thediversity (D) region genes, followed by the joining (J_(H)) region genesand the constant (C_(H)) gene cluster. The size of the locus isestimated to be about 2,500 kilobases (kb). During B-cell development,discontinuous gene segments from the germ line Ig H locus are juxtaposedby means of a physical rearrangement of the DNA.

Production of a functional heavy chain immunoglobulin polypeptiderequires three discontinuous DNA segments, from the V_(H), D, and J_(H)regions, to be joined in a specific sequential fashion generating thefunctional units. Once these units are formed specific heavy chains areproduced following transcription of the immunoglobulin locus. There aretwo loci for immunoglobulin light (Ig L)chains, the kappa locus on humanchromosome 2 and the lambda locus on human chromosome 22. The structureof the Ig L loci is similar to that of the Ig H locus, except that the Dregion is not present. The entire V region, or various fragments of theV region is used to produce a broad spectrum of high affinityantibodies. For example, a subset of the known V region genes of thehuman heavy and light chain Ig loci (Berman et al., EMBO J. 7: 727-738(1988)) is used to produce transgenic hosts, which transgenic host arecapable of mounting a strong immune response and provide high affinityantibodies. Antibodies or antibody analog producing B-cells from thetransgenic host are used, for example, for fusion to a mouse myeloidcell to produce hybridomas or immortalized by other conventionalprocess, i.e., transfection with oncogenes. These immortalized cells arethen grown, for example, in continuous culture or introduced into theperitoneum of a compatible host for production of ascites.

As discussed above, present invention also provides for the productionof polyclonal human anti-serum or human monoclonal antibodies orantibody analogs provided they retain the activities of the antibodiesof the invention. Epitope binding component of the present inventionrefers to proteins consisting of one or more polypeptides substantiallyencoded by genes of the immunoglobulin superfamily (i.e., TheImmunoglobulin Gene Superfamily, Williams & Barclay In: ImmunoglobulinGenes, Honjo, Alt, and Rabbitts, eds., (1989) incorporated herein byreference). For example, an epitope binding component comprises part orall of a heavy chain, part or all of a light chain, or both. However, anepitope binding component must contain a sufficient portion of animmunoglobulin superfamily gene product to retain the ability to bind toa specific target, or epitope.

Included within the scope of this invention is bispecific antibodiesthat are formed by joining two epitope binding components that havedifferent binding specificities.

It is well known that native forms of “mature” immunoglobulins varysomewhat in terms of length by deletions, substitutions, insertions oradditions of one or more amino acids in the sequences. Thus, both thevariable and constant regions are subject to substantial naturalmodification, yet are “substantially identical” and still capable ofretaining their respective activities.

Polynucleotides encoding human constant and variable regions areisolated in accordance with well known procedures from a variety ofhuman cells, but preferably immortalized B-cells. Similar methods areused to isolate non-human immunoglobulin sequences from non-humansources. Suitable source cells for the polynucleotides and theirexpressed and secreted products are obtained from a number of sources,such as the American Type Culture Collection (“Catalogue of Cell Linesand Hybridomas,” Fifth edition (1985) Rockville, Md., U.S.A.)

In addition to these naturally-occurring forms of immunoglobulin chains,“substantially identical” modified heavy and light chains are readilydesigned and manufactured utilizing various recombinant DNA techniqueswell known to those skilled in the art. For example, the chains varyfrom the naturally-occurring sequence at the primary structure level byseveral amino acid substitutions, terminal and intermediate additionsand deletions, and the like. Alternatively, polypeptide fragmentscomprising only a portion of the primary structure are produced, whichfragments possess one or more immunoglobulin activities (i.e., bindingactivity).

In particular, it is noted that like many genes, theimmunoglobulin-related genes contain separate functional regions, eachhaving one or more distinct biological activities. In general,modifications of the genes encoding the desired epitope bindingcomponents are readily accomplished by a variety of well-knowntechniques, such as site-directed mutagenesis (see, Gillman & Smith,Gene 8:81-97 (1979) and Roberts, et. al., Nature 328:731-734 (1987),both of which are incorporated herein by reference).

In preferred embodiments of the invention, the epitope binding componentof the antibody of this invention is encoded by immunoglobulin genesthat are “chimeric” or “humanized” (see, generally, Queen (1991) Nature351:501, which is incorporated herein by reference). Once expressed,VE-cadherin antibodies, epitope binding components, their dimers, orindividual light and heavy chains are purified according to standardprocedures of the art, for example, ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, Protein Purification, Springer-Verlag, N.Y. (1982)).Once purified, partially or to homogeneity as desired, the antibodiesand fragments thereof are then used, for example, therapeutically,diagnostically, in drug screening techniques, or in developing andperforming assay procedures, such as immunofluorescent stainings, andthe like.

Once a candidate anti-VE-cadherin monoclonal antibody is tested andconfirmed to have no increase in vascular permeability in vivo, the invivo activity and/or efficacy of the antibodies and antibody fragmentscan be determined by a number of methods know to those of skill in theart. Such assays include, but are not limited to, in vivo angiogenesisassays. Three in vivo angiogenesis assays, the corneal micropocket, theMatrigel plug and Alginate-encapsulated tumor cell assays areparticularly useful to assess anti-angiogenic activity of VE-cadherinmonoclonal antibodies. Typically, antibodies (or antibody fragments) arefirst tested in the corneal micropocket assay, since this assay requiresless antibody for testing and is less time-consuming than the otherassays. Various amounts of antibodies are either incorporated intosurgically implanted pellets or administered in a systemic manner. Thoseantibodies with significant inhibitory activity on cornealneovascularization are further tested in the Matrigel plug and Alginateassays. The Matrigel plug and Alginate assays serve to confirmanti-angiogenic activities of anti-VE-cadherin antibodies and allow forquantification of anti-angiogenic activity between various antibodiesand controls. Anti-VE-cadherin antibodies that show inhibition ofangiogenesis in vivo are further tested for their anti-tumor activity intumor models. A human A431 epidermoid carcinoma xenograft model, theLewis lung subcutaneous tumor model and the Lewis lung metastasis modelare used for these studies.

Example 5 provides additional assays as well as detailed examplesapplying such assays and techniques for further evaluating theantibodies and/or antibody fragments of the invention.

Pharmaceutical compositions comprising the antibody antagonists ofpresent invention are useful for administration to subjects in subjectsin need thereof. Administration is achieved by different routes ofadministration, including oral, or parenteral (subcutaneously,intramuscularly or intravenously.) The compositions for parenteraladministration commonly comprise a solution of the antibody or acocktail thereof dissolved in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers are used, i.e., water,buffered water, 0.4% saline, 0.3% glycine and the like. These solutionsare sterile and generally free of particulate matter. These compositionsare sterilized, for example, by conventional, well known sterilizationtechniques.

The carrier or diluent of the composition of invention comprises, forexample, pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. The concentration of the agent in these formulations varywidely, i.e., from less than about 0.01%, preferably at least about 0.1%to as much as about 5% by weight. The concentration range is selectedprimarily based on fluid volumes, viscosities, or particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intramuscular injectionis made up to contain, for example, about 1 ml sterile buffered water,and about 1 mg of the agent. A typical composition for intravenousinfusion is made up to contain, for example, about 250 ml of sterileRinger's solution, and 10 mg of the agent. Actual methods for preparingparenterally administrable compositions are known or apparent to thoseskilled in the art and are described in more detail in, for example, In:Remington's Pharmaceutical Science, 15th Ed., Mack Publishing Company(1980) which is incorporated herein by reference.

The antibodies of this invention are, for example, lyophilized forstorage and reconstituted in a suitable carrier prior to use. Thistechnique has been shown to be effective using conventionalimmunoglobulins and art-known lyophilization and reconstitutiontechniques. The compositions containing the present antibodies or acocktail thereof are administered for prophylactic and/or therapeutictreatments.

In accordance with the invention, a method of inhibiting angiogenesiscomprises administering a composition containing an antibody or antibodyfragment of the invention to a mammal for a time and in an amounteffective to inhibit angiogenesis. Similarly, the antibodies andantibody fragments can be used in methods of inhibiting tumor metastasisin a mammal by administering a composition containing an antibody of theinvention to a mammal for a time and in an amount effective to inhibitmetastasis of a tumor.

In one embodiment, the invention provides a method of treating a cellproliferative disorder associated with vascularization in a mammal whichcomprises administering a the composition containing an antibody orantibody fragment to a mammal in an amount effective to inhibitproliferation of endothelial cells without disturbing the normalvasculature.

Another embodiment relates to a method for reducing or inhibiting tumorvasculature in a mammal which comprises administering a compositioncontaining an antibody or antibody fragment to a mammal in an amounteffective to inhibit blood vessel formation without adversely affectingexisting vasculature. The tumors that can be treated in accordance withthe invention, include but are not limited to, carcinomas, gliomas,sarcomas, adenocarcinomas, adenosarcomas, adenomas as well as liquidtumors such as leukemic and lymphoid tumors. These tumors can occur inall parts of the body, for example, in the brain, breast, lung, colon,kidney, bladder, head and neck, ovary, prostate, pancreas, skin, bone,bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

As used herein “Cell proliferative disorders” refer to disorders whereinunwanted cell proliferation of one or more subset of cells in amulticellular organism occurs resulting in harm (i.e., discomfort ordecreased life expectancy) to the multicellular organism. Cellproliferative disorders occur in different types of animals and inhumans, and include blood vessel proliferative disorders, fibroticdisorders, angiogenesis, tumor growth, rheumatoid arthritis, andage-related muscular degeneration.

In another embodiment, the invention provides a method of gene therapywhich comprises administering a nucleic acid of encoding an antibody orantibody fragment of the invention to a mammal in an amount and for atime effective to inhibit angiogenesis at a predetermined site or toinhibit tumor neovascularization. Methods of gene therapy are known inthe art. This method is applicable to treating the diseases associatedwith angiogenesis as mentioned herein as well as for inhibiting thetumors listed above.

Therapeutic applications according to the invention, comprise treatmentprevention and amelioration. If treatment is intended, the compositionis administered to a patient already affected by the particular disease,in an amount sufficient to cure or at least partially arrest thecondition and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use depends upon the severity of the condition and the generalstate of the patient's own immune system, but generally range from about0.01 to about 100 mg of the antibody or antibody fragment per dose, withdosages of from about 1 to about 10 mg per patient being more commonlyused.

In prophylactic applications, compositions containing the antibodyantagonist, or a cocktail thereof if beneficial, is administered to apatient not already in a disease state to enhance the patient'sresistance. Such an amount is defined to be a “prophylacticallyeffective dose.” In this use, the precise amount again depends upon thepatient's state of health and general level of immunity, but generallyranges from about 0.1 to 100 mg per dose, preferably from about 1 toabout 10 mg per patient. Single or multiple administrations of thecompositions are carried out with dose levels and pattern being selectedby the treating physician. In any event, the pharmaceutical formulationsshould provide a quantity of the agent of this invention sufficient toeffectively treat the patient.

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed in an exemplary embodiment may be made by oneskilled in the art and it is intended that such modifications, changes,and substitutions are to be included within the scope of the presentinvention.

EXAMPLE 1 Methods

Monoclonal Antibody Preparation: Lewis rats (6-8 week old females) wereinjected subcutaneously (s.c.) with 0.1 ml of protein or peptide mixedin Freund's complete adjuvant using a 25-gauge needle. Rats were boostedevery 2-3 weeks with antigen and bled via the tail vein every week.After 3 booster immunizations or when sera titers reach maximal levels,mice were sacrificed by CO₂ inhalation. Spleens were recovered fromsacrificed animals for monoclonal antibody generation by conventionaltechniques.

Antibody Screening: Hybridoma supernatants were screened in by anenzyme-linked immunosorbent assay (ELISA) to identify antibodies whichbound to VE-cadherin.

Junction Formation/Ca Switch Assay: The junction formation assay wasdeveloped based on a modification of the calcium switch assay (Gumbiner,B., & Simons, K., Cell Biol. 102:457-468 (1986)). Transfectant CHO cellsor endothelial cells expressing VE-cadherin are plated onto glass slidesand allowed to form a confluent monolayer. The adherens junctions of themonolayer are artificially disrupted by depleting calcium from theculture medium by incubation with 5 mM EGTA for 30 min. EGTA-containingmedia is then removed and fresh media containing calcium is added to theculture to allow for formation of adherens junctions. The inhibition ofjunction formation is measured by addition of various concentrations ofanti-VE-cadherin monoclonal antibody at the time calcium-containingfresh media is added. The kinetics of junction disruption and junctionreformation processes correlate with the disappearance and reappearanceof VE-cadherin in the adherens junctions. The formation of adherensjunctions is visualized by immunofluorescent staining with a polyclonalantibody specific for mouse or human VE-cadherin. Immunostaining onanother junctional adhesion molecule (CD31) is routinely included toensure that the treated cell monolayer does not retract.

Paracellular Permeability Assay: The cell permeability assay isperformed by seeding VE-cadherin-expressing CHO cell transfectants orendothelial cells in the top chamber of Costar transwells. Cultures areincubated for 2 days to allow for formation of adherens junctions and aconfluent cell monolayer. Test antibodies are then added to the topchamber of cells along with FITC-dextran. The anti-VE-cadherin antibodyeffect on cell permeability (junction disruption) is measured as afunction of FITC-dextran that permeates into the bottom chamber.

The permeability assay can be adapted to a format useful for earlyscreening of monoclonal antibodies expressed in hybridoma culturesupernatants. In brief, hybridoma cells are seeded into the bottomchambers of transwells (Costar, 6.0 mm diameter/0.3 um pore size) andco-cultured with a monolayer of cells expressing mouse VE-cadherin onthe top filter. Cells used in this assay are either transfected CHOcells expressing the full-length mouse VE-cadherin molecule or the mouseH5V endothelioma cell line. After co-culture for 3-5 days, FITC-dextran(1 mg/ml) are added to the top chamber and permeability measured byfluorimetry as a function of FITC-dextran that crosses the cellmonolayer into the bottom chamber. Permeability activity (junctiondisruption) of candidate monoclonal antibody are calculated as thepercentage of increase in permeability of VE-cadherin expressing cellsas compared to control wells containing an unrelated control ratmonoclonal antibody. Permeability activity are normalized by hybridomacell counts and total rat IG production to control for variation ingrowth rate and antibody production between different hybridoma clones.The junction disrupting activity of the new monoclonal antibody arecompared to that of the monoclonal antibody 19E6, which is known to havehigh junction disrupting activity (>150% increase in permeability). Onlythose antibodies that exhibit no disruption (approx. 25% increase inpermeability) or modest disruption (approx. 25-75% increase inpermeability) activity are subjected to further screening for theirjunction inhibiting activity in the junction formation assay.

Corneal Pocket Assay: C57/BL mice (6-8 week old female) wereanesthetized with ketamine and a corneal pocket was created in both eyesusing a von Graefe cataract knife. Hydron pellets containing basic-FGFwith or without test antibody at various doses were then implanted intoeach eye pocket. Alternatively, hydron pellets containing basic-FGF wereimplanted and mice treated by i.p. injection with a 25-gauge needle oftest antibody at various doses or controls every 3 days. After 6-7 days,the angiogenic response was examined by slit-lamp biomicroscopy andphotographed. Mice were sacrificed by CO₂ inhalation and the eyesexcised and prepared for further histological analysis.

EXAMPLE 2 VE-Cadherin Monoclonal Antibodies that Inhibit AdherensJunction Formation without Disrupting Existing Junctions

Two groups of Lewis rats were immunized with either a mixture of fourKLH-coupled peptides having sequences from the N-terminal domain 1 ofmurine VE-cadherin (FIG. 2) or with affinity-purified soluble mouseVE-cadherin (smVEC-Ig) which had been expressed in CHO cells. Thisimmunogen encompasses the entire extracellular region of mouseVE-cadherin fused to human Fc chain. The resulting hybridoma clones weretested for production of antibodies with binding activity to VE-cadherinusing a conventional ELISA format. This screening identified twenty (20)rat anti-murine VE-cadherin antibodies, 10 from each of the originallyimmunized groups of rats.

Several properties of these monoclonal antibodies were examined and theresults are summarized in Tables 1 and 2.

The 20 candidate VE-cadherin antibodies were tested in the“calcium-switch” and “permeability” assays to examine their new junctionformation inhibiting activity and existing junction disrupting activity,respectively. Among these 20 antibodies, E4B9 was shown to specificallyinhibit adherens junction formation without adversely affecting normalvasculature (FIGS. 3 and 4). Furthermore, the E4B9 antibody was alsotested in an in vivo angiogenesis assay and showed greater than 80%inhibition of corneal neovascularization (FIG. 5). While anotherantibody (19E6) was also identified as a potent inhibitor ofVE-cadherin-mediated adherens junction formation by the in vitro assaycriteria, this antibody disrupts existing junctions (FIG. 3). The keybiological activities of these two antibodies are summarized in Table 3along with data from other murine and human anti-VE-cadherin antibodies.

TABLE 1 Anti-VE-Cadherin Antibodies Prepared Against Peptide ImmunogensBacterial Ca²⁺⁻switch Paracellular mECD1~2 Native VEC Assay PermeabilityMAb¹ (Blot) (Blot) (IF) (% Increase) 19E6² + + + 120~450 6D10 + + + 20E4B9 (P1)³ + + + <20 E4G10 (P1) + + + <20 E3F2 (P2) + − − <20 1F6.1(P2) + − − <20 10E4.1 (P2) + + − <20 8D6.1 (P4) + + − <20 9C6.1 (p4) + −− <20 3F7.1 (p4) + + − <20 4F1.1 Sup + + − <20 (mED1~2) ¹Abbreviations:MAb, monoclonal antibody; bacterial mECD1~2, bacterially-expressedprotein containing extracellular domains 1 and 2 of the N-terminus ofmurine VE-cadherin; IF, immunofluorescence. ²Control antibody. ³Thisantibody, E4B9, cross-reacts with human VE-cadherin.

TABLE 2 Anti-VE-Cadherin Antibodies Prepared Against smVEC-Ig ?? Ca²⁺⁻switch Paracellular MAb¹ ELISA (Blot) Assay (IF) Permeability Domain10G4 +++ + + + 1 9D9 + − − − 1 2G7 + − − − 1 13E6 +++ + − − 2 8A7 +++ +− − 2 5H6 ++ + − − 2 3C3 + − − − 2 15F12 +++ + − − 2 1A3 + − − − 2~32B11 +++ + − − 5 ¹See Table 1.

TABLE 3 Anti-human VEC Junction disruption vs. Junction inhibition MAb¹Epitope (Permeability Ca2+-switch) Cad5 Domain 1 +++ +++ BV9 Domain 3+++ +++ BV6 Domain 3 +++ +++ TEA Domain 4 +/− +/− Hec1.2 Domain 4 − −Anti-murine VEC Toxicity 19E6 Domain 1 +++ +++ + E4B9 Domain 1 +/− +++ −10G4 Domain 1 ND +++ ND 6D10 Domain 3-4 +/− +/− − ¹See Table 1.

EXAMPLE 3 E4B9 Cross reacts with Human VE-Cadherin

The murine epitope sequence recognized by antibody E4B9 shares 100%homology with human VE-cadherin, so this antibody was examined todetermine if it cross-reacts with human VE-cadherin. Western-blotanalysis of several VE-cadherin expressing human and murine cellindicated that E4B9 indeed cross-reacts with human VE-cadherin (FIG. 6).This finding facilitates development of a “humanized” E4B9 antibody andits success in the preclinical development since its anti-tumor activitycan be tested extensively in several mouse models.

EXAMPLE 4 Epitope Mapping

To define the specific VE-cadherin domain targeted by each newmonoclonal antibody, a series of smVE-cadherin-Ig truncations wererecombinantly generated. The epitope mapping strategy is shown in FIG.7. The culture supernatants from COS cells transfected with thesesmVE-cadherin-Ig truncation-bearing plasmids were used with ELISA todetermine the epitope domains for each monoclonal antibody. Fine epitopemapping of the three functional blocking monoclonal antibodies (E4B9,19E6 and 10G4) were made. The preliminary results showed that 19E6 and10G4 recognize regions different from that of monoclonal antibody E4B9(FIGS. 7-9).

Antibody E4B9 inhibits new junction formation without disruptingexisting junctions whereas other antibodies (19E6, 10G4 and Cad-5)disrupt existing junctions. During the later stage of angiogenesis,detached endothelial cells have to assemble into a capillary-liketubular structures that is mediated by the homophilic adhesion ofVE-cadherin molecules, presumably from the same cells (strand dimers)first and then from the opposing cells (adhesion dimers). Therefore, anantibody (such as E4B9) that antagonizes the “strand dimer” formation issufficient to inhibit new junction formation. In contrast, disruption ofthe existing junctions is a reversed process, i.e., from “adhesiondimers” to “strand dimers”. Those antibody antagonists that are specificto the “adhesion dimers” thus appear more disruptive to the existingvasculature. Evidence supporting this model is provided by fine epitopemapping and a crystal structure of VE-cadherin.

EXAMPLE 5 Miscellaneous In Vivo Assessments

Vascular permeability in tissues are analyzed by a Miles' type assaywith some modifications (Corda, et al., Proc. Natl. Acad. Sci.96:9815-9820 (1999) incorporated herein by reference. In brief, the testantibody or fragment is administered either intraperitoneally orintravenously to mice at various doses (50-1000 μg/dose). Increasedvascular permeability is determined by injecting Evans blue dye (100micro liter of 1 mg/ml) intravenously at various times (6 h, 12 h, 24 hand 48 h.) Following administration, typically 20 minutes later, miceare anesthetized by ketamine and perfused with approximately 20 ml ofPBS. Mouse organs are removed and homogenized in TCA/ethanol (1:1 v/v).The Evans blue content in the tissue homogenates are quantified byspectrophotometry (OD=510 nm). The antibody effect on vascularpermeability is measured as the percentage increase in Evans blue dyecompared to control antibody.

Anti-mouse VE-cadherin monoclonal antibodies are evaluated for theiranti-tumor effects in the Lewis lung subcutaneous primary tumor model,the Lewis lung metastasis model and the human epidermoid (A431)subcutaneous xenograft model. Primary subcutaneous Lewis lung tumors areestablished in C57BL/6 mice (6-8 week old females) by s.c. injection of1×10⁵ tumor cells in a suspension of Hanks balanced salt solution intothe right flank using a 22-gauge needle. Mice (10 mice/group) aretreated with VE-cadherin antibody (50-1000 μg) or an unrelated controlrat IgG every 3-4 days for 3-4 weeks or until the mice become moribund.Tumor volume is measured twice weekly using calipers and the volumecalculated using the formula-ÿ/6×diameter². Tumors from mice in eachtreatment group are removed surgically for histology and stained withanti-CD31 antibody to assess vascular density. In the Lewis lungmetastasis model, primary tumors are established in the footpads ofC57BL/6 mice. After 28 days, when the tumors reach approximately 100mm³, the primary tumor is removed and 24 h later mice (10 mice/group)are administered i.p. with VE-cadherin antibody (50-1000 μg) or anirrelevant control rat IgG every 3 days. After 4 weeks of treatment,mice are sacrificed and lungs examined for tumor metastasis. Lungs arealso examined by histology for evidence of micrometastases and stainedwith anti-CD31 antibody to assess vascular density.

The human epidermoid carcinoma cell line A431 is injected subcutaneouslyinto the right flank of a thymic mice. Once tumors reach 150 mm³, miceare divided randomly into treatment groups (10 mice/group) andadministered VE-cadherin antibody (50-1000 μg) or an unrelated controlrat IgG every 3 days for 4 weeks. Tumors are measured twice weekly andremoved after mice become moribund or at 4 weeks. Tumors are alsoexamined by histology and stained with anti-CD31 antibody to assessvascular density. Evaluation of the VE-cadherin therapy is based ontumor growth rate, tumor regression and histological evaluation ofneovascularization of tumors. The activity of the antibody in each tumormodel are compared to 19E6 monoclonal which serves as a positivecontrol. The negative control is an unrelated rat monoclonal antibody.Statistical analysis of tumor growth are determined using a two-tailedStudent's T-test where a p value of <0.05 is considered significant.

In Matrigel Plug Assay C57/BL mice (6-8 week old female) are injecteds.c. with 0.5 ml of angiogenic factors mixed in Matrigel using a25-gauge needle. Mice are then treated by i.p. injection with a 25-gaugeneedle with various doses of VE-cadherin antibodies or controls every 3days. After 10 days mice are sacrificed by CO2 inhalation and the plugsrecovered from the animals for further histological analysis.

In Agitate Encapsulated Tumor Cell Assay C57BL/6 mice (6-8 week oldfemale) are anesthetized with ketamine and then 4 beads surgicallyimplanted s.c. into the upper third of the back and pushed away from theincision site. The incision is closed with surgical clips. Mice are thentreated by i.p. injection with a 25-gauge needle with various doses ofVE-cadherin antibodies or controls every 3 days. After 12 days, mice areinjected i.v. with 100 ÿl of a 100 mg/kg FITC-Dextran solution(MW-150,000). Animals are sacrificed by C0₂ inhalation and beads areremoved, kept in the dark and processed for FITC quantitation.

In Human Tumor Xenograft Model, athymic nude (nu/nu) mice (6-8 week oldfemale) are injected s.c. with 2×106 A431 human epidermoid tumor cellsin a suspension of Hanks balanced salt solution into the right flankwith a 22-gauge needle. Once tumors reach 100-200 mm³ in size,VE-cadherin antibodies or a control antibody is administered to micetwice weekly by i.p. injection with a 25-gauge needle for 6 weeks oruntil mice become moribund. Tumor volumes are measured twice weekly withcalipers. Animals which become tumor free during the study are followedfor up to 8 weeks after the completion of treatment. Mice which completethe study or become moribund are then sacrificed by CO₂ inhalation.

1-22. (canceled)
 23. A method of inhibiting angiogenesis associated withan autoimmune disorder, collagenous vascular disease, rheumatoidarthritis, fibrotic disorders, or age-related muscular degeneration in amammal which comprises: administering a pharmaceutical composition to amammal having a condition selected from the group consisting of anautoimmune disorder, collagenous vascular disease, rheumatoid arthritis,a fibrotic disorder and age-related muscular degeneration for a time andin an amount effective to inhibit angiogenesis associated with thecondition, the pharmaceutical composition comprising: an isolatedmonoclonal antibody or fragment thereof capable of specifically bindingto a site on a mouse or human VE-cadherin, said site being within theabout first 15 N-terminal amino acids of domain 1 of the VE-cadherin,wherein said antibody or fragment thereof is capable of inhibitingVE-cadherin mediated adherens junction formation in vitro but does notexert any significant or substantial effect on paracellular permeabilityin vitro, and a pharmaceutically acceptable carrier or diluent.
 24. Themethod of claim 23, wherein the condition is rheumatoid arthritis. 25.The method of claim 23, wherein the condition is collagenous vasculardisease.
 26. The method of claim 23, wherein the condition is a fibroticdisorder.
 27. The method of claim 23, wherein the condition isage-related macular degeneration.
 28. The method of claim 23, whereinthe condition is an autoimmune disorder.
 29. The method of claim 23,wherein the antibody or fragment thereof does not exert any significantor substantial effect on vascular permeability in vivo.
 30. The methodof claim 23, wherein the antibody or fragment thereof inhibits formationof new adherens junctions without disturbing existing adherensjunctions.
 31. The method of claim 23, wherein the antibody or fragmentthereof is a single chain antibody, is humanized, is chimerized or isbispecific.
 32. The method of claim 23, wherein the antibody or fragmentthereof is fused to a heterologous polypeptide.
 33. A method of treatingrheumatoid arthritis which comprises: administering a pharmaceuticalcomposition to a mammal having rheumatoid arthritis, for a time and inan amount effective to inhibit proliferation of endothelial cellswithout disturbing normal vasculature, the pharmaceutical compositioncomprising: an isolated monoclonal antibody or fragment thereof capableof specifically binding to a site on a mouse or human VE-cadherin, saidsite being within the about first 15 N-terminal amino acids of domain 1of the VE-cadherin, wherein said antibody or fragment thereof is capableof inhibiting VE-cadherin mediated adherens junction formation in vitrobut does not exert any significant or substantial effect on paracellularpermeability in vitro, and a pharmaceutically acceptable carrier ordiluent, thereby inhibiting proliferation of endothelial cells withoutdisturbing normal vasculature in the treatment of rheumatoid arthritis.34. The method of claim 33, wherein the antibody or fragment thereofdoes not exert any significant or substantial effect on vascularpermeability in vivo.
 35. The method of claim 33, wherein the antibodyor fragment thereof inhibits formation of new adherens junctions withoutdisturbing existing adherens junctions.
 36. The method of claim 33,wherein the antibody or fragment thereof is a single chain antibody, ishumanized, is chimerized or is bispecific.
 37. The method of claim 33,wherein the antibody or fragment thereof is fused to a heterologouspolypeptide.